Human Ntn4 (Netrin 4) CLIA Kit (HUES01206)

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HUES01206
€599

Description

ELISA Kit Technical ManualMSDS

Human Ntn4 (Netrin 4) CLIA Kit

This kit uses Sandwich-CLIA as the method. The micro CLIA plate provided in this kit has been pre-coated with an antibody specific to Human Ntn4 . Standards or samples are added to the appropriate micro CLIA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human Ntn4 and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Human Ntn4, biotinylated detection antibody and Avidin-HRP conjugate will appear fluorescence. The Relative light unit (RLU) value is measured spectrophotometrically by the Chemiluminescence immunoassay analyzer. The RLU value is positively associated with the concentration of Human Ntn4. The concentration of Human Ntn4 in the samples can be calculated by comparing the RLU of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityHuman
Detection methodChemiluminescence
Detection range15.63-1000 pg/mL
Sensitivity9.38 pg/mL
Sample volume100uL
Sample typeSerum, plasma and other biological fluids
RepeatabilityCV < 15%

Specificity

This kit recognizes Human Ntn4 in samples. No significant cross-reactivity or interference between Human Ntn4 and analogues was observed.

Typical Data

As the RLU values of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.

Concentration
(pg/mL)
RLU Average Corrected
100051935
55415
5367553650
50021609
23369
2248922464
25011003
9419
1021110186
1254630
5172
49014876
62.52530
2376
24532428
31.251336
1226
12811256
15.63690
726
708683
025
25
25--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human Ntn4 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human Ntn4 were tested on 3 different plates, 20 replicates in each plate.

  Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (pg/mL)50.95150.70453.5255.38151.71459.89
Standard deviation6.4015.1650.434.7715.7550.54
C V (%)12.5610.0611.128.6110.3810.99

Recovery

The recovery of Human Ntn4 spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.

Sample Type Range (%) Average Recovery (%)
Serum (n=5)87-10192
EDTA plasma (n=5)91-10697
Cell culture media (n=5)91-10196

Linearity

Samples were spiked with high concentrations of Human Ntn4 and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.

    Serum (n=5) EDTA plasma (n=5) Cell culture media (n=5)
1:2Range (%)101-11884-9786-100
Average (%)1079193
1:4Range (%)99-115102-11687-101
Average (%)10511094
1:8Range (%)90-10299-11498-112
Average (%)97107104
1:16Range (%)99-11794-10797-111
Average (%)10899104

Kit Components & Storage

An unopened kit can be stored at 4'C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.

Item Specifications Storage
Micro CLIA Plate(Dismountable)8 wells X12 strips-20'C, 6 months
Reference Standard2 vials
Concentrated Biotinylated Detection Ab (100X)1 vial, 120 uL
Concentrated HRP Conjugate (100X)1 vial, 120 uL-20'C(shading light), 6 months
Reference Standard & Sample Diluent1 vial, 20 mL4'C, 6 months
Biotinylated Detection Ab Diluent1 vial, 14 mL
HRP Conjugate Diluent1 vial, 14 mL
Concentrated Wash Buffer (25X)1 vial, 30 mL
Substrate Reagent A1 vial, 5 mL4'C (shading light)
Substrate Reagent B1 vial, 5 mL4'C (shading light)
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 copy

Human Ntn4 (Netrin 4) CLIA Kit (HUES01206) Assay procedure

    1. Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
    2. Aliquot 100µl of standard solutions into the standard wells.
    3. Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
    4. Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids.) into test sample wells.
    5. Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
    6. Aspirate the liquid from each well, do not wash. Immediately add 100µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix.Incubate for 1 hour at 37°C.
    7. Aspirate or decant the solution from the plate and add 350µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
    8. Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
    9. Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
    10. Add 100µL of Substrate mixture solution to each well. Cover with a new plate seal andincubate for no more than 5 min at 37°C. Protect the plate from light.
    11. Determine the RLU value of each well immediately.

Human Ntn4 (Netrin 4) CLIA Kit (HUES01206) Protein Information

UniProt Protein Function:May play an important role in neural, kidney and vascular development. Promotes neurite elongation from olfactory bulb explants.
NCBI Summary:This gene encodes a member of the netrin family of proteins, which function in various biological processes including axon guidance, tumorogenesis, and angiogenesis. Netrins are laminin-related proteins that have an N-terminal laminin-type domain, epidermal growth factor-like repeat domain, and a positively charged heparin-binding domain at the C-terminus. The protein encoded by this gene is involved in processes including neurite growth and migration, angiogenesis and mural cell adhesion to endothelial cells. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Jul 2016]
UniProt Code:Q9HB63
NCBI GenInfo Identifier:1046552696
NCBI Gene ID:59277
NCBI Accession:NP_001316629.1
UniProt Secondary Accession:Q9HB63,Q658K9, Q7L3F1, Q7L9D6, Q7Z5B6, Q9BZP1, Q9NT44 Q9P133, B2RNC2,
UniProt Related Accession:Q9HB63
Molecular Weight:66,352 Da
NCBI Full Name:netrin-4 isoform 2
NCBI Synonym Full Names:netrin 4
NCBI Official Symbol:NTN4  
NCBI Official Synonym Symbols:PRO3091  
NCBI Protein Information:netrin-4
UniProt Protein Name:Netrin-4
UniProt Synonym Protein Names:Beta-netrin; Hepar-derived netrin-like protein
Protein Family:Netrin
UniProt Gene Name:NTN4  
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Additional Information

Product type:
ELISA
Reactivity:
Human
ELISA Type:
CLIA
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